HIV-1-associated pathogenesis in the immune system and central nervous system (CNS) is dependent on viral replication, which can be controlled by viral binding and entry, receptor/co-receptor usage, host cell proliferation and activation, cellular differentiation, antiretroviral therapy status, and regulation of viral expression by the long terminal repeat (LTR) in conjunction with viral and cellular regulatory proteins. Viral proteins that regulate LTR function and viral expression include the Tat trans-activator protein and viral protein R (Vpr), which can induce LTR activity through several cis-acting LTR elements. The replicative capacity of HIV-1 is further affected by evolution of the viral genotype (including tat, vpr, and the LTR) during the course of infection. The hypothesis of these investigations is that co-evolved viral regulatory elements (CVRE) Tat, Vpr, and the LTR alter the viral replicative phenotype, affect the spread of virus between compartmentalized cell types, and affect the course and severity of CNS disease. The specific aims of this application are to (1) establish a CVRE sequence database and a bank of DNA clones derived from HIV-1-infected immune cells (activated T cells, non-activated T cells, monocytic cells) and CNS-resident cells (microglia, perivascular macrophages, astrocytes) isolated from patients that differ in HAART and dementia status;(2) analyze the CVRE database for intrapatient variations (specific to cell type or compartmentalized to the CNS or immune system) and interpatient variations (HAART and dementia status-specific) in co-evolved Vpr, Tat, and LTR sequences;(3) construct infectious molecular clones (IMCs) incorporating select CVRE sequences and analyze each IMC-derived virus for CD4 binding, co-receptor utilization, and replication phenotype in immune- and CNS-derived cell lines and primary cells;and (4) determine the ability of co-evolved Tat-LTR, Vpr-LTR, and Tat-Vpr-LTR combinations from brain-derived CVRE clones to support transient expression, and establish correlations between transient LTR induction, dementia status, and HAART status. This unique approach to studying regulated HIV-1 expression in the context of CNS disease and HAART will provide greater insights into HIV-1-associated pathogenesis and may lead to more efficacious treatments of HIV-1 CNS disease.